YEAST: PRODUCTS AND DISCOVERY Proceedings of the 2 nd Australian Conference on Yeast: Products and Discovery 28-29 November 2002 Melbourne http://www.hsn.csiro.au/YPD/ CSIRO Health Sciences and Nutrition 343 Royal Parade, Parkville, Melbourne Editor: Dr. Paul R. Vaughan ISBN 0-9581991-0-8
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YEAST: PRODUCTS AND DISCOVERY
Proceedings of the 2nd Australian Conference on Yeast: Products and Discovery
28-29 November 2002 Melbourne
http://www.hsn.csiro.au/YPD/
CSIRO Health Sciences and Nutrition 343 Royal Parade, Parkville, Melbourne
Editor: Dr. Paul R. Vaughan
ISBN 0-9581991-0-8
Welcome to the 2nd Yeast: Products and Discovery Meeting
The photograph is of participants attending the 1st Yeast: Products and Discovery Meeting
at Couran Cove, Stradbroke Island, Queensland from 29 June- 1 July 2000
The organising committee would like to thank the following organisations for their
sponsorship and support of this meeting.
Australian Society of Biochemistry and Molecular Biology
http://www.asbmb.org.au/
Carlton and United Breweries
Commonwealth Scientific and Industrial Research Organisation
http://www.csiro.au/
YPD 2002 Conference Dinner Please note that if you wish to bring a parter/friend to the conference dinner you will need to submit a registration, paying only for the dinner (ie $35). Pre-Conference Mixer – REMINDER Carlton & United Breweries have kindly agreed to sponsor a YPD 2002 conference mixer at the CUB Brewhouse in Richmond on the eve of the conference,Wednesday November 27th from 6.00 - 8.00 pm. The Brewhouse is located on the corner of Nelson and Sth. Audley Streets (Melways ref. Map 2H, C1). There is parking available on Nelson St. immediately in front of the entrance and there is a regular tram service from the city. Tram 109 (to Mont Albert) can be picked up at tram stops anywhere along Collins St in the City, and ask to be dropped off at the first stop after Church St (Abbotsford). Sth. Audley St. is about 200 m past the tram stop (heading in the same direction as the tram). Alternatively a taxi from the City should not cost more than about $12 - $15. We can also arrange transport from the CBD or CSIRO, Parkville (the conference venue) for a limited number of registrants - but please let me know asap if you will require this. Please let Dr. Paul Chambers whether or not you will attend this mixer so that we can advise CUB on numbers.
Provisional Program (subject to change) * indicates a student presentation
Session Chair Speaker - Title Wednesday November 27, 2002
Mixer at the Brewhouse (sponsored by CUB) (6.00-8.00 pm)
Thursday November 28, 2002
Welcome (8.45 am)
Ian Macreadie
1. Genomics, proteomics and metabolomics 9.00 am-10.30 am
Paul Chambers tba *Anthony Heinrich - Comparative Proteomics: Identifying novel proteins in the winemaking strain Saccharomyces cerevisiae *Jeff Eglinton - Using metabolomics to investigate cellular metabolic changes in a glycerol overproducing strain of saccharomyces cerevisiae
Tea break and Poster Session
2. New yeast technology 11.00 am -12.30 pm
Rob Learmonth Rob Learmonth - Recent advances in laser scanning microscopy
*Carlos Rosado - Yeast autophagy: development of novel intracellular pH biosensors Mark Prescott - Applications for Fluorescent Proteins in yeast
Lunch break and Poster Session
3. Poster introductions 2.00 pm -3.30 pm
Those with posters will have an opportunity to give a short (5 minute) introduction.
Tea break and Poster Session
4. Wine yeast 4.00 pm -5.45 pm
Paul Henschke Graham Fleet – The grape berry as an ecosystem for yeasts and filamentous fungi Miguel de Barros Lopes - Investigation into the mechanism of action and biological role of Saccharomyces cerevisiae mannoproteins which reduce visible haziness in white wine Vladimir Jiranek – Strategies for avoiding problems associated with nitrogen limitation during wine fermentation Paul Grbin - Investigation of the formation of volatile phenols and fatty acids by Dekkera and Brettanomyces yeast *Kate Howell – Yeast strain dynamics during mixed culture wine fermentation and effect on wine composition *Simon Dillon – Can yeast strain affect colour and phenolic content of shiraz wine?
Conference Dinner at the Laureate from 6.30 pm (Wines sponsored by Univ. of Adelaide and Southcorp)
Thursday November 29, 2002
5. Yeast cell and molecular biology 9.00 am-10.30 am
Phil Nagley *Andrew Stephens - Yeast ATP synthase: protein engineering and topology *Onisha Patel - Using yeast as a model to demonstrate a new mechanism of sulfa drug action Ian Macreadie - Foreign gene expression in yeast
Tea break and Poster Session
6. Yeast stress Grant Stanley Meredith Chandler -
11.00 am -12.30 pm
tba Gabriel Perrone - Cellular functions in glutathione homeostasis identified by genome-wide screening / Engineering yeast for glutathione production
Lunch break and Poster Session
7. Brewing yeast 1.30 pm -3.00 pm
Peter J. Rogers (CUB)
Peter J Rogers (CUB) - New Variations on an age old profession: Peter L. Rogers (UNSW) - R&D opportunities in fuel ethanol production Dermot O'Donnell - Brewing yeasts-history, types and their applications
Tea break (take down posters)
8. Medical yeast and disease 3.15 pm. -4.45 pm
Paul Vaughan Whelan Meyer - Molecular epidemiology of C. neoformans revealing a possible link between the old and new world John Warmington - Will Genomics and Proteomics Produce the Next Generation of Antifungal Compounds? Peter Iliades - Modelling Pneumocystis carinii drug resistance in yeast
Ian Macreadie Presentations Paul Henshke Feedback and Discussion forum
LIST OF AUTHORS ALPHABETICAL with page number * Bold presenting
1. Beckhouse A.G., Higgins V.J., Rogers P. J., Dawes I.W. Ergosterol Requirement Of Yeast In The Protection Against Oxidative Stress In Industrial Fermentation
2. Jenny Bellon, Jeff Eglinton, Alan Pollnitz, Cherise Hillier, and Miguel de Barros Lopes
Hybrid Wine Yeasts With Unique Fermentation Characteristics 3. Shauna L. Brown, Miguel de Barros Lopes, Peter B. Høj, Elizabeth J. Waters
Investigation Into The Mechanism Of Action And Biological Role Of Saccharomyces cerevisiae Mannoproteins Which Reduce Visible Haziness In White Wine
4. B. A. Butcher and R. P. Learmonth
Yeast Membrane Responses To Glucose Are Related To Membrane Protein Expression. 5. M. Chandler, G. Stanley, P. Rogers, P. Chambers.
Gene Array Analysis Of Saccharomyces cerevisiae During Ethanol Stress. 6. Simon Dillon, Eveline Bartowsky, Peter Høj, Laurent Dulau, and Paul Henschke
Can Yeast Strain Affect Colour And Phenolic Content Of Shiraz Wine?
7. Jeff Eglinton and Paul Henschke
Saccharomyces Bayanus – An Alternative Wine Yeast? 8. Jeff Eglinton, Anthony Heinrich, Alan Pollnitz, Paul Henschke and Miguel de Barros Lopes
Using Metabolomics To Investigate Cellular Metabolic Changes In A Glycerol Overproducing Strain Of Saccharomyces Cerevisiae
9. G H Fleet, A L Beh, C. Prakitchaiwattana and G M Heard
The Grape Berry As An Eco-System For Yeasts And Filamentous Fungi 10. G.H. Fleet, A.L. Beh, C. Prakitchaiwattana, S.S. Bae, and G.M. Heard
The Yeast And Bacterial Ecology Of Wine Grapes 11. Jennie Gardner, Andrea Vystavelova, Miguel de Barros Lopes, and Vladimir Jiranek
Identification Of Genes Contributing To A “High Nitrogen Efficiency” (Hne) Phenotype In Yeast
Kinetic Analysis Of The Activation Of The One-Carbon Regulon Using Microarray Technology 13. E.M. Goldys, A. Donnelly, R. Wilkinson
Rapid Optical Essay For Identification Of Different Strains Of Baker’s Yeast Saccharomyces cerevisiae.
14. Paul R. Grbin, Mathew Reynolds, Peter Dillon, and Stephanie G. Lambert Investigation Of The Formaton Of Volatile Phenols And Fatty Acds By Dekkera And Brettanomyces
Yeast 15. Anthony Heinrich, Jelle Lahnstein, Kris Ferguson, Graeme Currie, Vladimir Jiranek and Miguel de
Barros Lopes.
Comparative Proteomics: Identifying Novel Proteins In The Winemaking Strain Saccharomyces cerevisiae.
16. L.Helfenbaum, C. McNees, C. Dyke, A. Lim, N. Amin, S. Shahin, R.Wickramasinga and M-J. Gething
Physiological Regulation of the Unfolded Protein Response in Yeast
17. V.J. Higgins, T. Oliver, P.J. Rogers and I.W. Dawes
Talking To Yeast: Application Of Genome-Wide Expression Analysis To Communicate Conditions Important For Successful Industrial Fermentations
The yeast Dekkera, the perfect form of Brettanomyces, when allowed to grow in wine can cause
a range of flavour modifications. These changes are generally considered to be undesirable.
Some of the sensory characters that these yeasts can produce in wine are attributed to volatile
phenols and fatty acids. The volatile phenols, 4-ethylphenol and 4-ethylguaiacol have been
described as; stable, farmyard, horsy and ‘band-aid’, while the fatty acid, iso-valeric acid has a
rancid cheese or vomit-like aroma.
A range of species and strains of Dekkera and Brettanomyces have been investigated for the
production of these aroma compounds. The yeast were grown in chemically defined media and
analysed by sensory evaluation and gas chromatography/mass spectrometry. The production of
the volatile phenols appears to be a unique characteristic of Dekkera, with only trace amounts or
none produced by the Brettanomyces species. The results confirmed that the precursor of 4-
ethylphenol is p-coumaric acid and that ferulic acid is the precursor of 4-ethylguaiacol. No
volatile phenols were produced in the absence of the phenolic acid compounds. All strains of
Dekkera investigated could produce iso-valeric acid. In the presence of exogenous L-leucine a
stimulation of iso-valeric acid was observed. A better understanding of the conditions that lead
to the formation of these flavour compounds will lead to the development of strategies to limit
their concentration in wine.
COMPARATIVE PROTEOMICS: IDENTIFYING NOVEL PROTEINS IN THE WINEMAKING
STRAIN SACCHAROMYCES CEREVISIAE.
Anthony Heinrich1,2,3, Jelle Lahnstein4, Kris Ferguson5, Graeme Currie5, Vladimir Jiranek2,3 and
Miguel de Barros Lopes1,3
1The Australian Wine Research Institute, Urrbrae, SA, 5064, Australia. 2Department of
Horticulture, Viticulture and Oenology, The University of Adelaide, PMB1, Glen Osmond, SA,
5064, Australia. 3Cooperative Research Centre for Viticulture, Urrbrae, SA, 5064, Australia. 4Department of Plant Science, Adelaide University, Urrbrae, SA, 5064, Australia. 5School of
Botany, University of Melbourne, Parkville, VIC, 3052, Australia.
Extensive phenotypic variation can exist between individuals of a single species. Elucidating the
genetic basis for this variation, especially in the post genome era, has received considerable
attention in view of its importance in to a broad range of objectives including predicting human
disease, defining biodiversity and improving agricultural yield. Saccharomyces cerevisiae wine
yeast strains have been selected over thousands of years of winemaking for properties that
include fast growth in high-sugar grape juices, high yield and tolerance to ethanol and, more
recently, sulfite resistance and the biosynthesis of flavour and aroma compounds at
concentrations beneficial for wine quality. However, the genes that contribute to these valuable
properties have not yet been elucidated. As ethanol is the main stress encountered during grape
juice fermentation, we have chosen to study how wine strains respond to an ethanol stress and
compare this to the response of less ethanol tolerant laboratory strains. By using a comparative
approach, we hope to establish which genetic factors play a role in the greater ethanol tolerance
of wine strains. A proteomic approach is being taken so that differences in protein modification
as well as expression can be monitored. The results have shown that many proteins are
differentially expressed in wine strains compared to laboratory strains. Proteins identified using
mass fingerprinting (MALDI-TOF) include glycolytic enzymes, proteins involved in amino acid
metabolism, and signaling proteins. However, limitations in the sensitivity of the current methods
and apparatus used to date and problems in comparing gels between experiments have made it
difficult to identify all proteins of interest. Our results achieved thus far and the difficulties
encountered with a comparative proteomics approach will be presented.
PHYSIOLOGICAL REGULATION OF THE UNFOLDED PROTEIN RESPONSE IN YEAST
L.Helfenbaum, C. McNees, C. Dyke, A. Lim, N. Amin, S. Shahin, R.Wickramasinga and M-J. Gething.
Department of Biochemistry and Molecular Biology, Russell Grimwade School of Biochemistry,
University of Melbourne, Parkville, 3010, Australia
The Unfolded Protein Response (UPR) is an intracellular signalling pathway between the endoplasmic
reticulum (ER) and the nucleus. The accumulation of unfolded proteins in the ER results in the
transcriptional upregulation of genes encoding ER resident chaperones and folding catalysts. In S.
cerevisiae, the ER to nucleus (ERN) signal transduction pathway is mediated by a sensor molecule Ern1p
which spans the ER membrane. UPR stress results in the oligomerisation and autophosphorylation of
Ern1p where upon it functions as an endoribonuclease splicing HAC1 mRNA encoding a b-ZIP
transcription factor. Hac1p recognises the unfolded protein response element (UPRE) found in the
promoters of genes transcriptionally upregulated in response to UPR stress.
We have shown that splicing of HAC1 mRNA is modulated by osmotic stress. Under conditions of low
osmolarity the cell integrity (CI) MAP Kinase module is activated in a Rho1p and protein kinase C
dependent manner. Deletion analysis of CI pathway components indicates that modulation of HAC1
mRNA splicing is primarily regulated through the action of PKC1 directly while the CI MAP Kinase
pathway is responsible for the level of accumulation of Hac1p. Furthermore we have demonstrated that a
pathway from the plasma-membrane receptors Wsc1p and Mid2p through the RhoGEF (GTP/GDP
Exchange Factor) Rom2p to Rho1p modulates the UPR.
The CI MAP Kinase pathway primarily mediates the cell integrity/cell proliferation response in yeast and
is activated in the cell-cycle at the stage of bud emergence. We propose that under conditions of cellular
stress during cell-wall remodelling the synthesis of cell wall components and plasma membrane is co-
ordinated by the interaction between the CI pathway and the UPR.
TALKING TO YEAST: APPLICATION OF GENOME-WIDE EXPRESSION ANALYSIS TO
COMMUNICATE CONDITIONS IMPORTANT FOR SUCCESSFUL INDUSTRIAL
FERMENTATIONS
V.J. Higgins1,2, T. Oliver3, P.J. Rogers3 and I.W. Dawes1,2
1Clive and Vera Ramaciotti Centre for Gene Function Analysis and 2School of Biotechnology
and Biomolecular Sciences, UNSW, Sydney, NSW, 3Carlton & United Breweries Ltd,
Abbotsford, VIC, Australia.
Industrial fermentation processes are dynamic systems that subject yeast to an ever-changing
range of stresses and differing metabolic requirements. However, the majority of research into
these processes is carried out using defined biological systems that do not always reflect the
true nature of the industrial process. To determine whether microarray technology could
produce a better understanding of environmental conditions important to industrial
fermentations, the genome-wide expression response of an industrial yeast strain during the
early stages of large-scale beer fermentation was analysed. During the first hour of fermentation
genes involved in ergosterol biosynthesis were induced. Cellular ergosterol levels were
measured and shown to correlate with the increased expression of the ergosterol biosynthetic
genes. Metabolic activity analysis of yeast mutants deficient in ergosterol biosynthesis showed
that its presence is essential for efficient adaptation to conditions of fermentation. Genes
involved in maintaining cell redox balance and protection against oxidative stress were also
induced during the initial stages of the fermentation. Many of these were part of the thioredoxin
and glutathione systems. This was a surprising result since the only aeration that occurred was
that caused by the agitation of the wort during the filling of the fermentation vessel. This amount
of aeration would not normally be regarded as a condition that would cause an oxidative stress
response in yeast cells. Genome-wide expression analysis was found to be an effective
communicator, through yeast gene expression profiles, of environmental conditions that are
important in industrial fermentations.
YEAST STRAIN DYNAMICS DURING MIXED CULTURE WINE FERMENTATION AND
During fermentation, yeast is subjected to range of stressful conditions; the major stressors
being ethanol, low pH, osmotic stress and nutrient limitation. Yeast stress during fermentation is
strongly associated with reduced growth rates and diminishing viability, leading to poor ethanol
yields and low productivity. An interesting development in studies on yeast stress is the
discovery that the adaptation rate to ethanol stress is improved significantly by the addition of
small quantities of acetaldehyde, however the biochemical and molecular processes
underpinning this effect are unknown. Identifying the mechanisms associated with the
acetaldehyde-mediated yeast response to ethanol stress may facilitate the production of yeast
strains with improved ethanol tolerance or the development of strategies for improving the
ethanol tolerance of yeast.
The presentation will focus on the level of gene expression in Saccharomyces cerevisiae during
ethanol stress in the presence and absence of acetaldehyde. Yeast were inoculated into fresh,
nutrient-rich medium alone, or medium with added acetaldehyde only, ethanol only or both
acetaldehyde and ethanol. Samples for biomass and RNA extraction were taken at regular
intervals during the initial growth lag period (ie. the period of adaptation to ethanol stress).
Growth curves showed a 65% increase in the stress adaptation rate in cultures containing both
ethanol and acetaldehyde compared to cultures containing ethanol only. Northern analysis was
conducted using probes specific for known ethanol-stress response genes and a housekeeping
gene.
CELLULAR FUNCTIONS IN GLUTATHIONE HOMEOSTASIS IDENTIFIED BY GENOME-WIDE
SCREENING / ENGINEERING YEAST FOR GLUTATHIONE PRODUCTION
Gabriel G. Perrone1, Chris M. Grant2 and Ian W. Dawes1
1 School of Biotechnology & Biomolecular Sciences, University of New South Wales, Sydney,
Australia; 2 School of Biochemistry, University of Manchester, Manchester, United Kingdom.
Glutathione is an essential metabolite protecting cells against oxidative stress and aging.
Genome-wide screening has identified 270 yeast deletion mutants that secrete significant
amounts of glutathione. These have identified a surprising set of functions that are important for
glutathione homeostasis. The highest secretors were affected in the late endosomal sorting
pathway which appears to play a crucial role in the balance between secretion and storage of
glutathione. Other functions included nitrogen/carbon source signalling, mitochondrial electron
transport, ion transport and those maintaining cellular integrity. These results have significant
implications for understanding mechanisms affecting glutathione depletion in degenerative
diseases and cell aging, and for engineering yeast cells to increase glutathione production.
STRATEGIES FOR AVOIDING PROBLEMS ASSOCIATED WITH NITROGEN LIMITATION
DURING WINE FERMENTATION
Kate Poole1,3, Jennie Gardner,1, Miguel de Barros Lopes2, and Vladimir Jiranek*, 1. 1Department of Horticulture, Viticulture and Oenology, The University of Adelaide, PMB 1, Glen
Osmond, SA 5064. 2Australian Wine Research Institute, PO Box 197, Glen Osmond, SA 5064. 3Present address: Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307, Dresden, Germany. *Corresponding author: Vladimir Jiranek,